7. Summary

Transcripción

7. Summary

Summary
7.
Summary
In the course of this work, three series of glycopeptides were synthesised. These three series
were analogous to the MUC1 repeating unit, which consists of twenty amino acids.
In the first section, a series of MUC1 glycopeptides was synthesised, based on new results
regarding the amino acid sequence of the repeating unit.
In the second section of this work, glycopeptides with N-glycosylation sites next to Oglycosylation sites were made. For examination of the synthesis, the sequence of the MUC1
repeating unit was chosen as a model.
In the third section of this work, glycopeptides bearing the sTF-antigen were synthesised
chemoenzymatically for the first time, using the catalysis of the 2,3-sialyltransferase.
The syntheses of the glycopeptides were performed manually, either in a multiple 20-hole
peptide synthesiser or on glass columns, the outlets of which were connected via a vacuum
manifold. Employment of the glycosyl amino acid building blocks 8, 25, 14 and 24 allowed
specific introduction of a monosaccharide unit (8, 25) or a disaccharide unit (14, 24) to the
growing peptide chain.
All of the glycopeptides synthesised here are intended to be tested in a T-cell stimulation
assay, and were presented to the laboratories of Prof. Dr. Joyce Taylor-Papadimitriou
(London) and Prof. Dr. Marianna Nuti (Rome) for execution of these tests. The parallel
investigations, which had already begun, were to be continued. In addition, Prof. Dr. HansGeorg Hanisch (Cologne) will also investigate the behaviour of the glycopeptides in the MHC
complex.
The following is a summary of the results of the individual sections:
1)
The amino acid sequence of the MUC1 repeating unit in cancer cells is subject to
polymorphism, whereas the MUC1 amino acid sequence of healthy cells is not. In the
cancerous MUC1 repeating units, the amino acids Asp8Thr9 are exchanged for Glu8Ser9, and
Pro19 is replaced with Ala19, Thr19 or Gln19 (Hanisch 2000). Bearing this in mind, the
glycopeptides 26–30 were synthesised in a manual 20-hole peptide synthesiser. The
glycosylation sites were introduced using monosaccharide building blocks 8 and 25. After the
successful synthesis of compounds 26–30, the MUC1 glycopeptides 36–45, modified with the
disaccharide building blocks 14 and 24, were produced. The proton shifts of the glycopeptides
26–30 and 36–45 could be assigned, using 1H1H-TOCSY and 1H1H-NOESY NMR spectra.
The sequences determined using NMR spectroscopy corresponded with the expected
glycopeptide sequences.
Summary
26
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
27
28
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala
29
30
AcO
AcO
OAc
O
OAc
O
AcO
AcO
AcNH
AcNH
Fmoc - Thr - OH
Fmoc - Ser - OH
25
8
36
37
38
39
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
40
41
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala
42
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Ser Ala Pro Pro Ala
43
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Ser Ala Pro Pro Ala
44
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Ser Ala Pro Pro Ala
45
AcO
OAc
O
AcO
OAc
O
O
AcO
AcO
OAc
O
AcO
OAc
O
O
AcO
AcNH
Fmoc -Thr - OH
14
AcO
AcO
AcNH
Fmoc -Ser - OH
24
Figure 7.1.: Glycopeptides 26–30 with the Tn monosaccharide building block 8 and 25, and glycopeptides 36–
45 with the TF disaccharide building block 14 and 24.
Summary
Glycopeptides 36–45 were tested using a T-cell stimulation assay in the laboratories of
Professor Joyce Taylor-Papadimitriou. In this assay, glycopeptide 38, which has a
glycosylation site at Ser5, resulted in stimulation which in a further test proved relatively
reproducible. Based on these results, glycopeptides 46–54 were synthesised for further
investigations. All glycopeptides 46–54 are glycosylated at Ser5; individual amino acids were
exchanged.
46
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala
47
48
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Gln Ala
49
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Gln Ala
50
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Thr Ala
51
Ala His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Thr Ala
52
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Ser Ala Pro Pro Ala
53
54
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Thr Ala
AcO
OAc
O
AcO
OAc
O
O
AcO
AcO
OAc
O
AcO
OAc
O
O
AcO
AcNH
Fmoc -Thr - OH
14
AcO
AcO
AcNH
Fmoc -Ser - OH
24
Figure 7.2.: Glycopeptides 46–54
2)
Investigations of a protein data bank (Swiss Prot) showed that exchange of Thr for
Asn in the DTR motif at the end of the tandem repeating domain in naturally occurring
MUC1 glycoproteins is possible. In addition, one finds N-glycosylation sites in MUC1
glycoproteins, between the repeating unit and the transmembrane domain. The synthesis of
mixed glycopeptides, which have a peptide chain containing both an N-glycosyl and an Oglycosyl carbohydrate side chain, is therefore interesting. In the course of this work, such
compounds (63–67), in which the peptide sequence is derived from the MUC1 structure, were
made for the first time.
For the syntheses, a new glycosyl amino acid building block was necessary, in order to
introduce a corresponding N-glycosidic carbohydrate side chain during the construction of the
glycopeptide. The glycosyl amino acid consisting of asparagine and chitobiose was chosen as
building block. The research group of Prof. Dr. Meyer already were experienced in the
synthesis of this compound.
Summary
While the coupling of the O-building blocks could be mostly conducted without any
problems, the coupling of the chitobiosyl building block was accompanied by the formation
of aspartamide as a side reaction.
The assignation of the proton shifts of glycopeptides 63–67, with sugar moieties at various
glycosylation sites, could be made using the 1H1H-TOCSY and 1H1H-NOESY NMR spectra.
The proton shifts of the carbohydrate protons of the various sugar residues could be assigned
using the 1H1H-TOCSY spectra.
63
Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
64
Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val
65
Ala His Gly Val Thr Asn Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
66
Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala Pro Gly Ser Thr Ala Pro Prö Ala
67
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Asn Ala
AcO
=
OAc
O
AcO
OAc
O
=
O
AcO
AcO
AcNH
Thr / Ser
14 (Thr) bzw . 24 (Ser)
AcO
AcO
OAc
O
O
AcNH
OAc
O
Asn
AcO
51
AcNH
Figure 7.3.: Glycopeptides 63–67 with N-glycosyl and O-glycosyl-linked carbohydrate side chains
3)
A cancer-associated reduction in expression of a glucosylaminoacidtransferase in
carcinoma cell lines leads to termination of the biosynthesis of the sugar side chains of
MUC1, so that the Tn monosaccharide and the TF monosaccharide appear in the side chain of
the MUC1 glycoprotein preferentially. These shortened sugar chains in turn serve as substrate
for specific sialyltransferases and this results in the formation of sialylated structures.
The purely chemical synthesis of a sialylglycosyl amino acid building block for a
corresponding glycopeptide synthesis is only possible via many reaction steps. The great
sensitivity and the negative charge of the 5-N-acetylneuraminic acid make a suitable
protective group combination necessary so that such a building block can be used for a
glycopeptide synthesis. A better alternative to organochemical synthesis is the enzymatic
transfer of the sialyl group, using CMP-NeuAc under catalysis of a sialyltransferase, onto a
complete glycopeptide, which has already been synthesised. In intial investigations, Kihlberg
was able to sialylate a synthetic MUC1 glycopeptide using a 2,3-sialyltransferase (Kihlberg
2001). Multiply glycosylated synthetic peptides have to date not yet been enzymatically
sialylated.
Summary
During this work, Neu5Ac was transfered from CMP-Neu5Ac to a series of multiply
glycosylated synthetic peptides bearing the TF antigen, under catalysis of 2,3sialyltransferase. Thus, compounds 76–83 could be achieved.
GalNAc-Gal-Neu5Ac
76
GalNAc-Gal-Neu5Ac
77
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
78
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
79
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
80
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
81
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
82
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
83
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
GalNAc-Gal-Neu5Ac
Figure 7.4.: Enzymatically sialylated glycopeptides 76–83
The 2,3-sialyltransferase necessary for this was expressed in HI5 insect cells by the research
group of Professor Henrik Clausen (Copenhagen), purified by treatment with Amberlite IRA95 and S-Sephadex, and concentrated by centrifugal filtration. The required glycopeptide
substrate was produced in a parallel solid phase synthesis on glass columns, and subsequently
sialylated with the 2,3-sialyltransferase. The reaction times of the enzymatic sialylation of the
glycopeptides with up to five glycosyl chains were always of a similar magnitude. Retardation
of the reaction by the glycosyl side chains already present was not observed. In the case of the
multiply sialylated compounds, a correspondingly larger amount of 2,3-sialytransferase and
CMP-Neu5Ac was required. An almost quantitative conversion could then always be
observed.
Summary
In the MALDI-TOF mass spectrometric investigation of the sialylated products 76–83,
fragmentation products arose, which resulted from the stepwise cleavage of one or more sialic
acid units. In the ESI mass spectra measured as a result of this, uniform molecular peaks
could be obtained. Analysis of the samples using reversed phase HPLC on a C-18 column
also indicate one uniform product. Upon closer examination of the 1H1H-TOCSY spectra,
however, several of the samples exhibit a weak, second carbohydrate trace. Thus, it is
possible that these trace substances, present in small amounts, may be incompletely sialylated
side products, which could not be fully separated from the products with the available
purification methods.

7. Summary

Transcripción

Documentos relacionados